Electrospray assisted capillary device for processing ultra low-volume samples

ABSTRACT

A spray-capillary device is configured to process ultra low-volume samples. The spray-capillary device includes a spray capillary that includes an inlet end and a discharge end. The spray capillary includes a porous section at the discharge end. A downstream connector provides an interface between the porous section of the spray capillary, a conductive fluid, and a high voltage electrical source. The application of voltage to the downstream connector causes electrospray ionization, which can be used to draw ultra law volume samples into the inlet end. A gas injection assembly can be used to increase the pressure on the inlet end of the spray capillary to encourage movement of the sample through the spray capillary. The spray-capillary device is well suited for providing ultra low samples to a mass spectrometer detection device.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/986,732 filed Mar. 8, 2020 entitled,“ELECTROSPRAY ASSISTED CAPILLARY DEVICE FOR PROCESSING ULTRA LOW-VOLUMESAMPLES,” the disclosure of which is herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract NumberAI141625 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND

There is interest, and practical value, in utilizing quantitativelow-volume sample techniques in mass spectrometry (MS) imaging andmicrosampling applications. The analysis of low-volume samples providesvaluable insight into complex biological systems. However, the proteomicand metabolomic analysis of low-volume samples remains challenging dueto the lack of simple, efficient, and reproducible microsamplingtechniques.

Efforts have been made to improve the sensitivity and throughput of lowquantity sample analysis in MS-based omics including the development ofspecialized sample preparation devices, high-resolution separationmethods, efficient spray-MS interfaces (i.e., novel ambient ionizationtechniques), and advancements in MS instrumentation. The sensitivity oflow quantity sample analysis has been dramatically improved using thesetechniques but many challenges remain in quantitative low-volume sampleinjection and extraction.

Micropipettes have been the most commonly applied tools for themanipulation of low-volume samples. Briefly, one end of the capillarytubing is pulled to make a micropipette to aspirate samples into thecapillary through a driving force. Two approaches have been applied asthe driving force for the operation of micropipettes: pump-basedextraction and electro-osmotic flow (EOF)-based extraction. In thepump-based micropipette extraction, a syringe is connected to a vacuumor mechanical pump to pull the sample into a capillary. Using thismethod, low sample injection volume can be accurately controlled.Coupling the pump-based extraction approach with offline capillaryelectrophoresis-MS for complex sample analysis has been used to studylive Xenopus laevis and Zebrafish embryos using a pump-basedmicropipette method. When an EOF-based method is utilized, an electrodeis inserted into a sampling capillary and current is applied to induceelectro-osmotic flow. Additionally, a micropipetting method based onelectro-osmotic injection was developed and applied to the analysis of asingle cell. During the collection process, +2 V was maintained toprevent the sample buffer from getting into the laser-pulled tips beforethe cell was penetrated. Low-volume cellular contents were extractedusing −2 V after penetration. This work demonstrated that EOF could beutilized as the driving force for microsampling low-volume samples suchas single onion cells. Another approach utilized an electro-osmotic pumpfor low-volume sample extraction from a Zebrafish embryo.

Other microsampling approaches include hydrodynamic methods, fluidicforce microscopy, capillary force, and electrowetting. A microfluidicchip-based platform, nanoPOTS, has been developed for low-volume sampleprocessing in single-cell proteomics analysis. NanoPOTS reduces totalprocessing volumes from the conventional hundreds of microliters to <200nL within a single droplet reactor. However, a customized automateddroplet-based microfluidic system must be incorporated for thelow-volume manipulation in nanoPOTS.

Continuous electrospray has been demonstrated using a platinum wireinserted into a laser pulled glass capillary with pre-injected samples.Additionally, researchers have proposed a sheathless interface togenerate continuous electrospray for MS detection.

As discussed above, numerous methods have been previously proposed forimproving microsampling techniques. While improvements have been made,current systems lack the ability to quantitatively manipulate ultralow-volume samples. The design of simple, efficient, reproduciblemicrosampling techniques have been a challenge. The currently availabledesigns result in considerable sample loss; require complicated, oftenproprietary equipment configurations; and cannot be directly coupledwith downstream separation techniques such as liquid chromatography (LC)and capillary electrophoresis (CE).

The novel electrospray-assisted device of the present disclosureaddresses the deficiencies of the previously proposed ultra low-volumesample extraction methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Several embodiments of the present disclosure are hereby illustrated inthe appended drawings. It is to be noted however, that the appendeddrawings only illustrate several embodiments and are therefore notintended to be considered limiting of the scope of the presentdisclosure.

FIG. 1 shows a schematic of the overall spray-capillary deviceconfigured for compound detection and/or separation.

FIG. 2 depicts the first connector, which is part of the gas injectionassembly.

FIG. 3 depicts the second connector, which is part of the of theconductive fluid injection assembly.

FIG. 4 shows a functional schematic of the overall spray-capillarydevice configured for compound detection and/or separation.

FIG. 5 shows a functional schematic of three steps for using thespray-capillary sample injection platform for MS detection.

FIG. 6A shows sample injection results and demonstrated reproducibilityfor run-to-run.

FIG. 6B shows sample injection results and demonstrated reproducibilityfor day-to-day.

FIG. 6C shows sample injection results and demonstrated reproducibilityfor batch-to-batch sample injections.

FIG. 7 shows a sample injection flow rate estimation using acamera-monitoring approach.

FIG. 8 shows the effect of vacuum force on the spray-capillaryperformance.

FIG. 9 shows (A) the effect of the viscosity of column liquid on thespray-capillary performance, and (B) the injection flow rated plottedagainst the viscosity for the three types of column liquid used.

FIG. 10 shows quantitative results of a spray-capillary device coupledwith MS for sample injections. (A) EICs of AngII (m/z=523.77−523.80)were evaluated with different injection times. (B) Calibration curve wasconstructed as a function of injection time (N=3 for each experimentcondition). (C) The injection reproducibility was demonstrated hereusing three replicated injections with 60 s injection times using theEICs of AngII (m/z=523.77−523.80).

FIG. 11A shows an evaluation of spray-capillary performance as afunction of capillary inner diameter.

FIG. 11B shows the injection reproducibility of low-volume sample at nLlevel sample (˜2.9 nL) using a spray-capillary device coupled to MS.

FIG. 11C shows an evaluation of spray-capillary performance as afunction of capillary length

FIG. 11D shows an evaluation of spray-capillary performance as afunction of electrospray voltage.

FIG. 12 shows an evaluation of the influence of random injection force(e.g., capillary action or sample adherence to the sample inlet surface)and the EOF effect on the spray capillary injection.

FIG. 13A shows a calibration curve of standard peptides separation usingthe spray-capillary CE-MS platform. (N=3 for each experiment condition).

FIG. 13B shows the reproducibility of peptide separation using thespray-capillary CE-MS platform. Sample injection time is 60 s for allreplicates.

FIG. 13C shows the EICs of Syn-2 (m/z=503.30−503.37) with differentsample injection times.

FIG. 13D shows the EICs of AngII (m/z=523.77−523.80) with differentsample injection times.

FIG. 14 shows evaluation of the detection limit of the spray-capillarysample injection for CE-MS. (A) An example of online peptide separationof Syn-2 and AngII (3 kV ESI voltage and 5 s sample injection time). (B)Calculated detection limits of Syn-2 an AngII based on the EICs usingequation indicated above figure.

DETAILED DESCRIPTION

The analysis of ultra low-volume samples provides valuable insight intocomplex biological systems. However, the proteomic and metabolomicanalysis of such samples has remained challenging due to the lack ofsimple, efficient, and reproducible microsampling techniques. Thepresent disclosure is directed to an electrospray-assisted(“spray-capillary”) device for quantitative low-volume sampleextraction. The spray-capillary device enables reproducible andquantitative microsampling with low injection flow rates (e.g., as lowas 15 pL/s). Stable electrospray is achieved through a chemically etchedtip from a long (e.g., 50 cm) capillary with a conductive sheath flow.The results can be replicated with capillaries longer than 50 cm. Thismethod is effective for capillaries between 10 cm to 200 cm. Thiselectrospray provides the driving force to quantitatively drawlow-volume samples into the capillary. Unlike prior technologies, thespray-capillary device can be directly coupled with capillaryelectrophoresis (CE) and/or other liquid chromatography separationtechniques without any additional device(s). The spray-capillary devicecan be used, for example, for high-throughput quantitative omicsanalysis of ultra low-volume samples such as single cells.

Before describing various embodiments of the present disclosure in moredetail by way of exemplary description, examples, and results, it is tobe understood that the present disclosure is not limited in applicationto the details of methods and compositions as set forth in the followingdescription. The present disclosure is capable of other embodiments orof being practiced or carried out in various ways. As such, the languageused herein is intended to be given the broadest possible scope andmeaning; and the embodiments are meant to be exemplary, not exhaustive.Also, it is to be understood that the phraseology and terminologyemployed herein is for the purpose of description and should not beregarded as limiting unless otherwise indicated as so. Moreover, in thefollowing detailed description, numerous specific details are set forthin order to provide a more thorough understanding of the disclosure.However, it will be apparent to a person having ordinary skill in theart that the embodiments of the present disclosure may be practicedwithout these specific details. In other instances, features which arewell known to persons of ordinary skill in the art have not beendescribed in detail to avoid unnecessary complication of thedescription.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those having ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which the present disclosure pertains. Allpatents, published patent applications, and non-patent publicationsreferenced in any portion of this application are herein expresslyincorporated by reference in their entirety to the same extent as ifeach individual patent or publication was specifically and individuallyindicated to be incorporated by reference.

As utilized in accordance with the methods and compositions of thepresent disclosure, the following terms, unless otherwise indicated,shall be understood to have the following meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or when the alternatives are mutually exclusive,although the disclosure supports a definition that refers to onlyalternatives and “and/or.” The use of the term “at least one” will beunderstood to include one as well as any quantity more than one,including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 100, or any integer inclusive therein. The term “at least one”may extend up to 100 or 1000 or more, depending on the term to which itis attached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y and Z.

As used herein, all numerical values or ranges include fractions of thevalues and integers within such ranges and fractions of the integerswithin such ranges unless the context clearly indicates otherwise. Thus,to illustrate, reference to a numerical range, such as 1-10 includes 1,2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc.,and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., upto and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2,2.3, 2.4, 2.5, etc., and so forth. Reference to a series of rangesincludes ranges which combine the values of the boundaries of differentranges within the series. Thus, to illustrate reference to a series ofranges, for example, a range of 1-1,000 includes, for example, 1-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200,200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, and includesranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000. The range 100units to 2000 units therefore refers to and includes all values orranges of values of the units, and fractions of the values of the unitsand integers within said range, including for example, but not limitedto 100 units to 1000 units, 100 units to 500 units, 200 units to 1000units, 300 units to 1500 units, 400 units to 2000 units, 500 units to2000 units, 500 units to 1000 units, 250 units to 1750 units, 250 unitsto 1200 units, 750 units to 2000 units, 150 units to 1500 units, 100units to 1250 units, and 800 units to 1200 units. Any two values withinthe range of about 100 units to about 2000 units therefore can be usedto set the lower and upper boundaries of a range in accordance with theembodiments of the present disclosure.

A reference to fractions of liters, such as 1 pL to 100 μL is intendedto explicitly include all quantities in the range, such as 10 pL to 1μL. Where used herein, the term ultra low-volume sample refers to asample having a volume in a range of 15 pL to 25 nL.

As used herein, the words “comprising” (and any form of comprising, suchas “comprise” and “comprises”), “having” (and any form of having, suchas “have” and “has”), “including” (and any form of including, such as“includes” and “include”) or “containing” (and any form of containing,such as “contains” and “contain”) are inclusive or open-ended and do notexclude additional, unrecited elements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Throughout this application, the terms “about” and “approximately” areused to indicate that a value includes the inherent variation of error.Further, in this detailed description, each numerical value (e.g.,temperature or time) should be read once as modified by the term “about”(unless already expressly so modified), and then read again as not somodified unless otherwise indicated in context. As noted, any rangelisted or described herein is intended to include, implicitly orexplicitly, any number within the range, particularly all integers,including the end points, and is to be considered as having been sostated. For example, “a range from 1 to 10” is to be read as indicatingeach possible number, particularly integers, along the continuum betweenabout 1 and about 10. Thus, even if specific data points within therange, or even no data points within the range, are explicitlyidentified or specifically referred to, it is to be understood that anydata points within the range are to be considered to have beenspecified, and that the inventors possessed knowledge of the entirerange and the points within the range. The use of the term “about” maymean a range including ±10% of the subsequent number unless otherwisestated.

As used herein, the term “substantially” means that the subsequentlydescribed parameter, event, or circumstance completely occurs or thatthe subsequently described parameter, event, or circumstance occurs to agreat extent or degree. For example, the term “substantially” means thatthe subsequently described parameter, event, or circumstance occurs atleast 90% of the time, or at least 91%, or at least 92%, or at least93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%,or at least 98%, or at least 99%, of the time, or means that thedimension or measurement is within at least 90%, or at least 91%, or atleast 92%, or at least 93%, or at least 94%, or at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99%, of thereferenced dimension or measurement (e.g., length).

As used herein any reference to “one embodiment” or “an embodiment”means that a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearances of the phrase “in one embodiment” in variousplaces in the specification are not necessarily all referring to thesame embodiment.

As used herein any reference to “we” as a pronoun herein refersgenerally to laboratory personnel or other contributors who assisted inthe laboratory procedures and data collection and is not intended torepresent an inventorship role by said laboratory personnel or othercontributors in any subject matter disclosed herein.

Turning to FIG. 1, shown therein is a depiction of one embodiment of aspray-capillary device 100. In this embodiment, the spray-capillarydevice 100 employs capillary electrophoresis (CE) and electrosprayionization (ESI) to drive low-volume samples from a container 200 to adetection device 202, such as a mass spectrometer (MS). The container200 can include a sample, background electrolytes (BGE), column liquids,buffer liquids, or other fluids intended to be processed through thespray capillary 102. As illustrated in FIGS. 4 and 5, it will beunderstood that multiple containers 200 may be involved in the operationof the spray-capillary device 100. The spray-capillary device 100includes a spray capillary 102, a gas injection assembly 104, a liquidinjection assembly 106, and a voltage generator assembly 108.

The spray capillary 102 includes a porous section 110 at the dischargeend 112, which may also be referred to as the “MS end.” The poroussection 110 acts as a spray nozzle through the discharge end 112. Thespray capillary 102 also includes an inlet end 114 on the opposite endof the spray capillary 102 from the discharge end 112. The inlet end 114may also be referred to as the “sample end.” The inlet end 114 can beplaced into the container 200. The preparation of the spray capillary102, and in particular the preparation of the porous section 110, isdescribed in detail below.

The liquid injection assembly 106 includes a downstream connector 116, asource of conductive liquid 118, a conductive liquid pump 120, and aliquid injection line 122. The liquid pump 120 can be a syringe, a smallpump, or an arrangement in which gravity forces the conductive fluid tomove from the source of conductive liquid 118 to the downstreamconnector 116 through the liquid injection line 122. As betterillustrated in FIG. 2, the downstream connector 116 includes a firstbranch 124, a second branch 126, and a third branch 128. In exemplaryembodiments, the downstream connector 116 is configured as “tee”connector that is manufactured from a suitable polymer, such as PEEK.The spray capillary 102 enters the downstream connector 116 at the firstbranch 124 and exits the downstream connector 116 through the secondbranch 126.

The porous section 110 of the spray capillary 102 extends from insidethe downstream connector 116 through the second branch 126 toward thedischarge end 112. In this way, conductive liquid inside the downstreamconnector 116 is permitted to enter the spray capillary 102 through theporous section 110. A metal sleeve 130 can be positioned over, oradjacent to, a portion of the porous section 110 to impose an electricalcharge on the fluids within the spray capillary 102. The metal sleeve130 is an example of a “conductive interface” on the downstreamconnector 116.

The gas injection assembly 104 includes an upstream connector 132, asource of pressurized gas 134, and a gas injection line 136, and a gastransfer line 138. As better illustrated in FIG. 3, the upstreamconnector 132 includes a first branch 140, a second branch 142, and athird branch 144. In exemplary embodiments, the upstream connector 132is configured as “tee” connector that is manufactured from a suitablepolymer, such as PEEK. The gas injection line 136 extends from thesource of pressurized gas 134 to the third branch 144 of the upstreamconnector 132. The gas can be nitrogen or another inert gas. The gas iscarried from the upstream connector 132 to the container 200 through thegas transfer line 138, which is secured to the first branch 140 of theupstream connector 132. In exemplary embodiments, the gas transfer line138 connects to the container 200 in a sealed manner to prevent therelease of pressure within the container 200. In certain embodiments, asdepicted in Step 1 of FIG. 5, it may not be necessary to use the gasinjection assembly 104, or to only use the gas injection assembly 104for a portion of the sample processing operation.

In exemplary embodiments, the inlet end 114 of the spray capillary 102is immersed within a fluid in the container 200. The spray capillary 102then passes in a coaxial manner inside the gas transfer line 138 throughthe first branch 140 and second branch 142 of the upstream connector132. The spray capillary 102 extends from the second branch 142 of theupstream connector 133 into the first branch 124 of the downstreamconnector 116, where the porous section 110 is positioned to admitconductive liquid entering the downstream connector 116 from the liquidinjection assembly 106. The spray capillary 102 exits the downstreamconnector 116 through the second branch 126 and the metal sleeve 130.The discharge end 112 of the spray capillary 102 is optimally located inclose proximity to the detection device 202.

The voltage generator assembly 108 includes the metal sleeve 130, avoltage source 146, a first lead 148, a second lead 150, and an upstreamlead 152. The voltage source 146 provides a suitable electricalpotential. The first lead 148 connects a first side (either positive ornegative) of the voltage source 146 to the first branch 140 of theupstream connector 132. The first lead 148 is connected to the upstreamlead 152, which extends from the first branch 140 to the container 200.In exemplary embodiments, the upstream lead 152 extends inside the gastransfer line 138. The second lead 150 connects the second side of thevoltage source 146 (oppositely charged from the first side) to thedownstream connector 116. In exemplary embodiments, the second lead 150is connected between the second side of the voltage source 146 and themetal sleeve 130. In this way, an ionizing voltage can be applied tofluid in the spray capillary 102 from the inlet end 114 in the container200 to the portion of the spray capillary 102 extending through themetal sleeve 130 in accordance with established electrospray ionization(ESI) principles. It will be appreciated that FIGS. 1-3 are not drawn toscale and are merely intended to provide a functional description of thevarious components within the spray-capillary device 100.

During the ESI process, a liquid jet is created when the electrostaticforce of the column liquid overcomes the surface tension, which reducesthe pressure around the discharge end 112 (spray tip) and creates apressure difference between opposing ends of the spray capillary 102.The pressure difference varies based on the experimental set-up, but canbe estimated using Poiseuille's Law. For example, for a 50 cm, 50 μminner diameter spray-capillary device, the difference is 0.068 psi when4 kV is used as the voltage and water is used as the column liquid.

The spray-capillary device 100 utilizes this pressure difference toserve as the driving force to move the liquid inside the spray capillary102 towards the discharge end 112. The conductive porous section 110 wasutilized to generate ESI as the driving force to quantitatively drawlow-volume samples into the long capillary (e.g., 50 cm). FIGS. 4 and 5show the spray-capillary device 100 platform and detailed diagram of thesheathless CE-MS interface with the detection device 202. Furthermore,the sample inlet end 114 of the spray capillary 102 can be directlyinserted into a background electrolyte (BGE) solution after sampleinjection for online CE-MS analysis, which further increases thehigh-throughput omics analysis of ultra low-volume samples.

In some embodiments, the gas injection assembly 104 is usedsimultaneously with the electrospray ionization (ESI) made possible bythe liquid injection assembly 106 and voltage generator assembly 108. Inother embodiments, the electrospray ionization function begins beforeactivation of the gas injection assembly 104, but is later disabled oncethe gas injection assembly 104 has been activated. In other embodiments,the gas injection assembly 104 is used before activation of theelectrospray ionization function. In yet other embodiments, theelectrospray ionization is used in isolation (as illustrated in Step 1of FIG. 5), while in other embodiments the gas injection assembly 104 isused in isolation. In yet other embodiments, the gas injection assembly104 and electrospray ionization function are activated and deactivatedmultiple times during a sampling process.

The present disclosure is directed to ultra low-volume sampling methodfor use with mass spectroscopy. The application of the microsamplingmethod of the present disclosure is not limited to mass spectroscopydetection. The disclosed sampling method an be utilized for ultralow-volume and low-volume microsampling analyzed with other detectionand imaging methods. The device and method disclosed, in one embodiment,is configured to be directly coupled with a MS as the CE-MS platformused for the analysis of chemical compounds, biochemical on biologicalsamples (i.e., tissues, saliva samples, serum samples etc.), theanalysis of single cells, ultra low-volume samples, and the detectionand analysis of chemicals (e.g., metabolites, proteins and peptides) insolvent mixtures or low volume fractions from other separation methodssuch as nano-LC.

EXAMPLES

The present disclosure will now be discussed in terms of severalspecific, non-limiting, examples. The examples described below, whichinclude particular embodiments, will serve to illustrate the practice ofthe present disclosure, it being understood that the particulars shownare by way of example and for purposes of illustrative discussion ofparticular embodiments of the present disclosure only and are presentedin the cause of providing what is believed to be a useful and readilyunderstood description of construction procedures as well as of theprinciples and conceptual aspects of the inventive concepts.

Example 1—Sample Injection

In at least one embodiment, the spray-capillary device 100 is configuredto perform quantitatively-controlled, evenly drawn microsamples, fromultra low-volumes. One non-limiting example of how the disclosed deviceis fabricated is by using the spray capillary 102,commercially-available sample vials 200, and standard electric wire forthe first lead 148, second lead 150 and upstream lead 152 (see FIGS. 1and 4-5). The chemicals, reagents and capillaries used in thedevelopment of the spray-capillary device 100 include, in onenon-limiting embodiment, Angiotensin II (AngII, A9525), Syntide 2(Syn-2, SCP0250), HPLC water (270733), ACS-reagent Acetonitrile (ACN,360457), formic acid (FA) (≥95%, F0507), and hydrofluoric acid (HF,≥48%, 30107) purchased from Sigma-Aldrich. Fused-silica capillaries werepurchased from Polymicro Technologies. AngII and Syn-2 stock solutionswere prepared in HPLC water. The standard peptide mixture used in thedescribed embodiment was a solution of 10 μM AngII and 10 μM Syn-2 (0.1%FA in 45% ACN in water).

The spray capillary 102 may be modified by a similar technique used tofabricate some sheathless interfaces, but the design parameters of thedisclosed spray-capillary device are modified specifically to achievequantitative control for microsampling applications. One of the keypoints in the design and development of the spray capillary design isthe porous section 110 which allows for the creation of the electrosprayas a stable and controllable pulling source for quantitatively handlingultra low-volume samples. In addition, a sample pulled into thespray-capillary can be directly analyzed without additional unionsconnected to it, which results in decreased sample loss. The tip of theporous section 110 is shaped specifically for microsampling, so thateven single cell samples can be retrieved.

Spray-Capillary Device Design

To produce precise, clean and reproducible interfaces, both thedischarge end 112 and sample inlet end 114 of the spray capillary 102,were cut using a Shortix™ capillary column cutter, and were evaluatedusing an inverted microscope. Briefly, the outside polymer coating ofthe discharge end 112 (about 3 cm) was removed by flame. The exposedsilica was etched using a 49% HF solution at room temperature togenerate the porous section 110 for electric contact. During the etchingprocess, the spray capillary 102 was continuously flushed with water ata flow rate of 0.2-0.4 μL/min to prevent etching of the inner wall bythe HF solution.

After etching, the width of the spray capillary 102 wall wasapproximately 5 μm in one embodiment. The tip shape and porous conditionof the porous section 110 were evaluated using an inverted microscope.The discharge end 112 of the spray capillary 102 was inserted into thedownstream connector 116 through the metal sleeve 130, which wasconstructed from stainless steel (4 cm, 1/16″ O.D., 0.04″ I.D.) so thatsome portion of the porous section 110 (e.g., about 1.5 cm) emerged fromthe metal sleeve 130. A continuous flow of conductive liquid (0.1% FA, 1μL/min) driven by the syringe pump 120 was introduced into the thirdbranch 128 of the downstream connector 116 to create the electriccontact for generating ESI. The ESI voltage was applied to the stainlesssteel metal sleeve 130 through an alligator clip. In the experimentalset-up of this embodiment, a sheathless interface is used to apply thevoltage to the metal sleeve 130. The second lead 150 electrical wire isconnected to the high voltage supply 146 with an alligator clipconnected to a screw which interacts with a metal conductive unit, a nutin this example, to transmit the voltage signal from the power supply tothe metal sleeve 130, as shown in FIGS. 1 and 4-5. The sample inlet end114 of the spray capillary 102 was either placed directly into a samplevial 200 for sample injection, as seen in steps 1 and 2 of FIG. 5, orinto a BGE vial 200 using the second upstream connector 132, shown inStep 3 of FIG. 5. As explained above, the second upstream connector 132of FIG. 2 has a gas inlet (third branch 144) to help control the flowrate, and the upstream lead 152, a metal wire in this example, is usedto apply high voltages for CE separation. The upstream lead 152 is heldin place by a gasket, and is extended into the BGE vial 200 adjacent tothe tip of the inlet end 114 that is also immersed in the BGE solution.The voltage is applied to the upstream connector 132 from the electricalsource 146 via an alligator clip attached to the metal sleeve 130extending perpendicular to the downstream connector 116. The powersource used for the BGE solution is separate, or at least requires aseparate output, from the power supply 146 used for the downstreamconnector 116 of the spray-capillary device 100.

Development and Characterization of the Spray-Capillary Device

The spray-capillary device 100 embodiment described herein can beutilized to generate continuous ESI. For example, before sampleinjection, the sample inlet end 114 of the spray-capillary was placed inthe sample vial 200 with the conductive liquid (0.1% FA). Low-pressurenitrogen was introduced into the sample vial 200, via the upstreamconnector 132 shown in FIG. 2, to fill the spray capillary 102 a dropletwas observed at the discharge end 112. Then the sample inlet end 114 ofthe long 102 capillary was immersed in the sample vial 200 with standardpeptide samples; the sample injection process was initiated byapplication of high voltage on the downstream connector 116 to generatecontinuous ESI as shown in FIGS. 1 and 4-5. The sample-injection flowrate was monitored and recorded using a digital camera. A segment of thepolymer coating on the sample inlet end 114 of the spray capillary 102was removed by flame so that the spray capillary 102 was transparent,and the contents could be visually observed. For the proof-of-principleexperiments, H₂O was used as the column liquid and an organic solution(90% n-butanol) was used as sample so a clear boundary could beobserved. The sample injection flow rate was calculated by recording themigration time of the sample/column liquid interface movement in thespray capillary 102 as seen in FIG. 7.

A 50 cm spray-capillary (360 μm O.D.×50 μm I.D.) was used forproof-of-concept experiments, although the length of the capillary tubeis not limited to this length. Initially, to determine the primarydriving force for sample injection into the spray-capillary device, agrounded copper plate was used as the spray target instead of the MSinlet to eliminate the vacuum force from the MS inlet capillary of thedetection device 202. The sample injection flow rate was estimated usingthe aforementioned monitoring method (H₂O as the column liquid). Theresults, shown in FIG. 14, indicate that ESI can be used alone as thedriving force for sample injection, with an injection flow rate of 160.7pL/s. The vacuum of the inlet of the detection device 202 can increasethe sample injection flow rate to about 263.8 pL/s. However, the vacuumfrom the MS inlet alone does not initiate the injection procedure asthere was no obvious movement of the sample boundary observed under thecamera.

The reproducibility of sample injection was further evaluated using thesame spray-capillary and using reproductions. The 50 cm spray-capillary(360 μm O.D.×50 μm I.D.) was used with the injection voltage of 4 kV.The 50 cm capillary length and the 4 kV injection voltage are notlimiting parameters. The length of the spray capillary 102 can bebetween 10 cm to 200 cm and the working injection voltage is 2.6 kV to 4kV. Four individual sample injections were evaluated using the samecapillary and conditions. The average sample injection flow rate in thiscapillary was estimated to be 255.2 pL/s and an RSD value of 4.9% wasobtained demonstrating reasonable reproducibility was evaluated amongruns as seen in FIG. 6A. Similarly, the day-to-day reproducibility andcalculated an RSD of 3.29% shown in FIG. 6B. Triplicate experiments werealso performed using two different spray-capillaries 102 to determinebatch-to-batch reproducibility, which yielded an RSD value of 5.8% shownin FIG. 6C.

The effect of the viscosity of the column liquid on injection flow ratewas also tested to evaluate the influence of friction on liquid motionin the capillary. For evaluation, three types of column liquids withdifferent viscosities (H₂O, 50% ACN, 90% n-butanol, theoreticalviscosities listed in FIG. 9) were evaluated using the spray-capillarydevice 100; injection flow rates were calculated. Samples were chosen sothat a clear boundary could be observed between the column liquid andthe sample shown in FIG. 9(A). The results in FIG. 5(B) suggested aninverse proportionality between flow rate and viscosity such that highersample injection flow rates were observed for low viscosity columnliquids. The increase in friction of the higher viscosity liquid mayresult in slower liquid motion, as suggested by Poiseuille's law.

Other factors that may affect the spray-capillary injection process suchas EOF, capillary action, and sample adherence to the surface wereevaluated based on the following set of experiments (FIG. 12). (1)Background experiments (evaluation of random sample injection such ascapillary action or sample adherence to the surface): the sample inletend of spray-capillary was inserted into sample vials (10 s, 30 s, 60 s,N=3) with no voltage applied; (2) EOF experiments (evaluation of theeffect of EOF on sampling): the sample inlet end of the spray-capillarywas inserted into sample vials (10 s, 30 s, 60 s, N=3) with theapplication of 100 and 500 V. These voltage values were chosen becauseno electrospray is formed under these conditions. This experiment canprovide us with a quantitative measurement of EOF during the injection,if any EOF exists. The capillary action or sample adherence to thesurface does contribute a relatively small amount of the sample that isinjected into the capillary during the spray-capillary process (lessthan 10%). The EOF effect on the tip contributes minimally to the sampleinjection process. Overall, the results suggest that ESI is the maindriving force for the spray-capillary experiments and EOF by itself doesnot significantly affect the sample aspiration.

Example 2—Sample Injection and MS Detection

In another embodiment, the present disclosure provides characterizationof the spray-capillary device 100 coupled with MS detection. Forexample, experiments determined the feasibility of the spray capillary102 to be directly coupled with MS for quantitative sample injection andanalysis. After the sample injection, a pressure-based sample elutionstep was incorporated to flush the sample through the spray capillary102 for MS detection. Briefly, nitrogen (˜2.5 psi) was introduced into aglass sample vial 200 filled with column liquid (0.1% FA in 45% ACN)using the pressure-based upstream connector 132 (FIG. 2). For MSdetection, 2.3˜2.6 kV was used for the electrospray ionization on themetal sleeve 130 of the sheathless interface of the downstream connector116.

A 100 μM solution of AngII (0.1% FA in 45% ACN) was used to characterizethe quantitative microsampling ability using the spray-capillary device100 (360 μm O.D., 50 μm I.D., 50 cm in length) when coupled with MSdetection. Sample was injected into the spray-capillary 102 using an ESIvoltage of 4 kV for 2, 15, 30, 60, and 90 s (FIG. 6(A)). An injectiontime correlation plot (FIG. 10(B)) was constructed based on theintegrated peak areas of extracted ion chromatograms (EICs) of AngII 2+ions. Good linearity (R²=0.98) indicates the method is quantifiable fora wide range of injection times. The total injection volume ranges from520 pL to 23.7 nL, which was estimated based on our pre-calculatedcapillary flow rates (˜260 pL/s) from camera monitoring. We alsoevaluated the reproducibility of the spray-capillary device 100 coupledwith MS detection. Triplicate experiments with 60 s injection time weredemonstrated in FIG. 10(C) with an average RSD value of 5.79%, which iscomparable to the previous camera-based evaluation.

Pressure-Based Sample Elution Setup and Fabrication

After injection of the sample into the spray-capillary 102, the gasinjection assembly 104 was utilized to apply pressure-based sampleelution for the follow-up MS detection. Nitrogen from the source ofpressurized gas 134 was injected through the gas injection line 136,through the third branch 144 into the upstream connector 132, where itwas carried by the gas transfer line 138 into the sample vial 200.Pressure in the sample vial 200 was consequently generated. The sampleinlet end 114 of the spray-capillary 102 was inserted into the glasssample vial 200 with a rubber cap to seal the vial inlet when pressurewas applied. The increased pressure within the sample vial 200encouraged the movement of the sample inside the spray capillary 102towards the discharge end 112. A gas gauge (0-3 psi) was used to controlnitrogen pressure for sample elution.

Mass Spectrometry Analysis

An LTQ Orbitrap Elite mass spectrometer was utilized for relatedspray-capillary experiments. The temperature of the inlet capillary was275° C. For pressure elution experiments, both 2+ and 3+ AngII ions weretargeted with two scan ranges: 523-527 m/z for 2+ ions, and 348-352 m/zfor 3+ ions. Both scans were acquired at the resolving power of 120,000at m/z=400. All data files were collected in profile mode. Peakextraction was done in XCalibur using RAW files. Results were processedand plotted using GraphPad Prism. In addition, chromatograms extractedfrom raw data were processed using boxcar smoothing algorithms.

System Parameters

Several other parameters can be tuned to vary the rate of sampleinjection, such as capillary inner diameter, capillary length, and ESIvoltage. (1) Capillary inner diameter: we evaluated the performance ofthe spray-capillary device 100 with a 50 cm length spray capillary 102with 20 μm, 50 μm, and 75 μm I.D. The sample injection time was 60 s forall spray capillaries 102. The results suggested that the sampleinjection volume increased as the spray-capillary inner diameterincreased (FIG. 11A). FIG. 11B shows a relatively high reproducibility(RSD=11.3%) of detected MS signals for spray-capillary injections with20 μm I.D. The sample injection rate was calculated using thespray-capillary with 20 μm I.D. using camera monitoring. The estimatedinjection rate is 15.0 pL/s without vacuum and 48.2 pL/s when thespray-capillary was placed in front of the inlet to the MS detectiondevice 202. Some discontinuity in the peak shape and deviations fromlinearity was observed as the spray-capillary parameters were varied,which may be cause by the fluctuations of the N₂ source. The flowresistance at the interface between the sample and running buffer underpressure may also contribute to some of the observed variation. (2)Spray-capillary length: Spray-capillaries 102 (360 μm O.D.×50 μm I.D.)with different lengths (30 cm, 40 cm, and 50 cm) were used to determinethe effect of spray-capillary length on injection volume. To minimizepotential errors, the same spray-capillary 102 was used for all theexperiments by trimming the capillary from the sample inlet end 114 toproduce the desired capillary lengths while the same MS discharge end112 was used. The strength of the MS signal decreased as the length ofthe spray-capillary increased, FIG. 11C. This inverse trend is likelydue to the result of the longer plug of the column liquid present inlonger capillaries, which leads to a stronger friction force and lowerinjection flow rate. (3) ESI voltage: A 50 cm spray-capillary with 360μm O.D.×50 μm I.D. was used to perform a series of experiments atvarying electrospray voltages (between 2.6 kV and 4.5 kV) (FIG. 7D). 2.6kV was the lowest ESI voltage determined to produce stable electrospraywith the 50 μm I.D. spray-capillary. 4.5 kV was the highest ESI voltageselected because arcing started to affect the ESI process when thevoltage was raised beyond this limit. The 2.6 kV and 4.5 kV voltages arenot limiting parameters for other possible embodiments since anembodiment with a different detection system than presently describedcan produce stable electrospray results under different ESI voltagesthan those presently described. A linear relationship was found betweeninjection volume and electrospray voltage. The formation of the cone-jetdepends on the balance between the surface tension of the sprayingliquid and the electric field force. Therefore, a linear relationshipbetween sample injection volume and ESI voltage is expected because,when electrospray voltage increases, the electric field force increasesas well, resulting in a higher sample injection rate.

Example 3—Sample Injection and Peptide Separation

In another embodiment, the present disclosure provides a new method ofdirect coupling of the spray capillary 102 with a CE-MS platform. Oneadvantage of the spray-capillary device 100 is its ability to directlyserve as the CE separation column after sample injection. Thespray-capillary device 100 can be directly coupled to the MS detectiondevice 202 to produce an online spray-capillary CE-MS platform. Wetested the performance of the spray-capillary CE-MS using a standardpeptide mixture (10 μM AngII and 10 μM Syn-2). A bare spray-capillary(50 cm in length, 360 μm O.D., 50 μm I.D.) was used for theseexperiments. For sample injection, the ESI voltage was set at 3 kV, andthe sample injection time was varied from 5 to 60 s (N=3 for eachcondition). Baseline separation was achieved for these two peptidesunder these conditions. Reasonably good reproducibility was observed forboth CE-MS elution time (RSD=9.3%) and extract ion intensities ofindividual peptides (RSD=6.64% for Syn-2 and RSD=9.71% for AngII) shownin FIGS. 13A-D. In addition, a good linear relationship between detectedsignals and sample injection time were detected for both AngII (R²=0.93)and Syn-2 (R²=0.97), indicating that spray-capillary 102 is capable ofquantitative sample injection when coupled with the CE-MS platform shownin FIG. 13A. The reproducibility of peptide separation using thespray-capillary CE-MS platform, and an injection time of 60 s, is shownin FIG. 13B. The results of EICs of Syn-2 (m/z=503.30-503.37) withdifferent injection times is shown in FIG. 13C. The results of EICs ofAngII (m/z=523.77-523.80) with different injection times is shown isFIG. 13D.

In the present work, the calculated theoretical plate number ofAngiotensin II was approximately 30,000, which is lower than recent CEstudies using the same peptide (˜300,000). To improve the separationresolution for complex sample analysis, several improvements to the CEseparation can be made. For example, experiments that utilize a longerspray capillary 102 with a smaller inner diameter combined with higherapplied voltages have resulted in higher sensitivity and resolutionseparations of peptides and metabolites. These experiments have alsodemonstrated utility when the sample is limited, such as in the analysisof single cells. In addition, high-quality capillary coatings, such aslinear polyacrylamide (LPA), have been used to improve separation powerby eliminating EOF. The performance of the spray-capillary device 100may be evaluated and improved using the modifications described above toachieve better quantitative results.

The detection limit for reproducible sample injection volume wasestimated based on data collected using 3 kV ESI voltage and 5 s sampleinjection. The sample injection flow rate (57.8 pL/s) was estimatedusing the previously described video-monitoring approach. For the 3 kVand 5 s sample injection, the total injected sample volume was 290 pL,and the injection amount of each peptide was about 2.9 fmol. Theestimated average detection limits were calculated through extrapolationand were found to be 17 amole and 65 amole for Syn-2 and AngII,respectively shown in FIG. 14. The highly reproducible separationperformance of the standard peptide mixture indicates thespray-capillary device 100 can be a useful tool to analyze complexbiological samples with optimized CE conditions such as longer columnsor coated capillaries.

CE Separation of Standard Peptide Mixtures

In a non-limiting embodiment, the spray-capillary device 100 can becoupled directly with a CE-MS system 202 to separate standard peptides.A spray-capillary (360 μm O.D., 50 μm I.D., 50 cm in length) was usedfor the CE separation of standard peptide mixtures. The MS discharge end112 of the spray-capillary 102, with a 3 cm porous section 110, wasinserted into the sheathless interface (downstream connector 116), asdescribed above for spray-capillary-based sample injection. Then thesample inlet end 114 was inserted into the sample vial 200. After sampleinjection, the sample inlet end 114 of the spray-capillary 102 wasinserted into a container 200 with the BGE solution (0.1% FA). The CEseparation was then conducted by applying 15 kV (300 V/cm) at the sampleinlet end 114. The 15 kV utilized for this experiment is not a limitingparameter. The working range can be 10-30 kV depending on the length ofcapillary utilized and the exact experimental configuration. Highvoltage (HV) from the power supply 146 was applied to the sampleinjector (upstream connector 132) using an alligator clip connected tothe upstream lead 152 immersed in the BGE in the vial 200. The upstreamconnector 132 was constructed as depicted in FIG. 2. Stainless-steelcomponents are not a limiting requirement of the disclosed method andsystem, the method and system works using components that are conductiveand configured to carry a voltage signal. The described examples andembodiments utilize stainless-steel components due to the costeffective, non-oxidizing nature (copper, an oxidizing conductor can alsobe used). After a 15 min CE separation, N₂ was introduced by the gasinjection assembly 104 at low pressure, typically 0 psi-3 psi withtypical operating pressure around 1-2 psi, to elute peptides. 2.6 kV wasused for the electrospray ionization at the sheathless interface. The2.6 kV utilized for these experiments is not a limiting parameter. Theexpected normal operating range is 1.5 kV-5 kV depending on the specificdetection system utilized. For the CE-MS experiments, full MS scans wereacquired at the resolving power of 120,000 at m/z=400, and the AGCtarget was set as 1E6 with the maximum ion injection time of 1000milliseconds (ms). The scan range is 150-2000 m/z.

In another non-limiting embodiment, the spray-capillary device 100 ischaracterized offline, monitored with a high resolution digital monitor.This embodiment can be replicated with or without a vacuum force tovalidate the results.

Example 4—Single Cell Sampling

In another non-limiting embodiment, the embodiment described in Example1 can be replicated using a sharp tip at the sample inlet end 114 of thespray capillary 102. This tip of the inlet end 114 can be sized toaccommodate samples of only a single cell. The sampling system andmethod of the present disclosure is designed to be adjustable and thecomponents interchangeable to accomplish a number of non-proprietary labsetups. The result is low cost experimental methods for quantitative,ultra low sample applications.

While the present disclosure has been described in connection withcertain embodiments so that aspects thereof may be more fully understoodand appreciated, it is not intended that the present disclosure belimited to these particular embodiments. On the contrary, it is intendedthat all alternatives, modifications and equivalents are included withinthe scope of the present disclosure. Thus the examples described above,which include particular embodiments, will serve to illustrate thepractice of the present disclosure, it being understood that theparticulars shown are by way of example and for purposes of illustrativediscussion of particular embodiments only and are presented in the causeof providing what is believed to be the most useful and readilyunderstood description of procedures as well as of the principles andconceptual aspects of the presently disclosed methods and compositions.Changes may be made in the structures of the various componentsdescribed herein, or the methods described herein without departing fromthe spirit and scope of the present disclosure.

It is claimed:
 1. An apparatus for measuring an ultra low-volume sample,comprising: (1) a spray capillary, wherein the spray capillary comprisesan inlet end having a non-porous tip, positioned to receive the ultralow-volume sample from a container 200, and a tapered discharge endhaving a porous section, wherein the spray capillary 102 is configuredto contain the ultra low-volume sample, wherein the porous section isconfigured to exert an electric force on the spray capillary; (2) anupstream connector comprising a first branch, a second branch, a thirdbranch, and an upstream lead conductor, wherein the spray capillary ispositioned within the upstream connector, extending from the firstbranch through the second branch, wherein the non-porous tip of thefirst inlet end of the spray capillary is positioned to receive theultra low-volume sample, wherein the upstream lead conductor ispositioned adjacent to the non-porous tip of the inlet end of the spraycapillary and extends perpendicular to the upstream connector at thefirst branch, and wherein the third branch is configured to receive agas; (3) a downstream connector comprising a first branch, a secondbranch, a third branch, and a conductive interface, wherein the tapereddischarge end of the spray capillary is positioned within the downstreamconnector such that the tapered discharge end of the spray capillary isinserted into the first branch and exits from the second branch, whereinthe third branch 128 of the downstream connector inlet is configured toreceive a conductive liquid from the source of conductive liquid suchthat the conductive liquid surrounds the tapered and porous dischargeend of the spray capillary, and wherein the conductive interface isconfigured for contact with the conductive substance, and wherein theconductive interface is configured to receive an electric signal; andwherein, responsive to the conductive interface of the downstreamconnector receiving the electric signal, the conductive interface formsa pressure differential between the inlet end of the spray capillary andthe tapered discharge end of the spray capillary.
 2. The apparatus ofclaim 1, wherein the first connector is configured with the second inletpositioned perpendicular to the first inlet and the first output end ofthe first connector; and wherein the second connector is configured withthe fourth inlet positioned perpendicular to the third inlet and thesecond output end of the second connector.
 3. The apparatus of claim 1,wherein the capillary is constructed of fused silica.
 4. The apparatusof claim 1, wherein the capillary is fabricated as a sheathlessinterface comprising an outer polymer coating which extends to theporous tip, which is free of the outer polymer coating.
 5. The apparatusof claim 1, wherein the capillary has a length in a range between 10 cmand 200 cm.
 6. The apparatus of claim 1, wherein the conductor issecured with a gasket, positioned at the first inlet of the firstconnector.
 7. The apparatus of claim 1, wherein the gas is nitrogen. 8.A method for processing an ultra low-volume sample, comprising the stepsof: providing spray-capillary device, comprising: (1) a spray capillary,wherein, the spray capillary comprises an inlet end having a non-poroustip, positioned to receive the ultra low-volume sample, and a tapereddischarge end with a porous section, the spray capillary configured tocontain the ultra low-volume sample, wherein the tapered discharge endis configured to exert an electric force, wherein upon receiving theelectric force; the tapered discharge end is configured to discharge anelectrospray, wherein upon discharge of the electrospray, a pressuredifferential is exerted on the spray capillary configured to contain theultra low-volume sample; (2) a first upstream connector comprising afirst branch, a second branch, a third branch, and an upstream leadconductor, wherein the spray capillary is positioned within the upstreamconnector, extending from the first branch through the second branch,wherein the non-porous tip of the inlet end of the spray capillary ispositioned to receive the ultra low-volume sample and enter the inletend, wherein the upstream lead conductor is positioned adjacent to thenon-porous tip of the inlet end of the spray capillary, and wherein thethird branch is configured to receive a gas; (3) a second downstreamconnector comprising a first branch, a second branch, a third branch,and a conductive interface, wherein the tapered discharge end of thespray capillary is inserted into the first branch of the downstreamconnector and exits out of the second branch of the downstreamconnector, wherein the third branch of the downstream connector isconfigured to receive a conductive substance that surrounds the poroussection discharge end of the spray capillary, and wherein the conductiveinterface is configured to receive an electric signal; introducing theultra low-volume sample into the inlet end of the spray capillary;injecting the conductive substance into the third branch of thedownstream connector, wherein the conductive substance passes out of thesecond branch of the downstream connector; and applying the electricsignal to the conductive interface of the downstream connector forforming the pressure differential between the inlet end of the spraycapillary and the tapered discharge end of the spray capillary; therebyforming the electrospray, causing the ultra low-volume sample to beinjected into the inlet end the spray capillary.
 9. The method of claim8 comprising introducing the electrospray into an analytic detectiondevice for analysis of the ultra low-volume sample.
 10. The method ofclaim 8, further comprising applying a vacuum to an analytic detectiondevice assembly after introducing the ultra low-volume sample into thespray capillary.
 11. The method of claim 8, wherein the electric signalis applied to the downstream connector to provide a voltage in a rangebetween 1.5 kV and 5 kV.
 12. The method of claim 8, wherein the upstreamconnector is configured with the third branch is positionedperpendicular to the first branch and the second branch of the upstreamconnector; and wherein the downstream connector is configured with thethird branch positioned perpendicular to the first and second branchesof the downstream connector.
 13. The method of claim 8, wherein thespray capillary is constructed of fused silica.
 14. The method of claim8, wherein the spray capillary has an inner sheathless interface and anouter polymer coating which extends to the porous section, which is freeof the outer polymer coating.
 15. The method of claim 8, wherein thespray capillary has a length in a range between 30 cm and 100 cm. 16.The wherein the conductor is secured with a gasket, positioned at thefirst branch of the upstream connector.
 17. The method of claim 8,wherein the spray capillary has an inner diameter in a range between 20μm and 75 μm.
 18. The method of claim 8, wherein the inlet end of thespray capillary is configured to retrieve a single cell sample.
 19. Themethod of claim 8, further comprising separating one or more peptides.20. The method of claim 19, wherein the step of separating the one ormore peptides comprises: inserting the inlet end into an electrolytesolution; and applying a voltage signal to the conductor, wherein theconductor is positioned in the electrolyte solution.